1. Field of the Invention
This invention relates to a hemoglobin based composition and the method to make the same. In particular, it relates to a method for crosslinking a hemoglobin based solution to create a hemoglobin based composition which has the capacity to transport oxygen for an increased length of time, while still retaining at least the oxygen affinity of hemoglobin in human red cells.
2. Description of the Prior Art
In current medical practice, when it is necessary to infuse patients who have experienced blood loss, such as trauma victims or surgical patients, with oxygen-carrying materials, only whole blood or packed, red blood cells are used. It is necessary to carefully match the donor and the recipient blood type; testing which can delay the blood infusion. As a result, patients suffering substantial blood loss are subjected to periods of oxygen deprivation which is detrimental. Furthermore, even when autologous, patient-donated, red blood cells are available through previous phlebotomy and storage, the oxygen-carrying capacity and safety of these autologous cells has declined as a consequence of the storage. As a result, for a period of as much as 24 hours after transfusion, the patient may be subject to sub-optimal oxygen delivery. Finally, there is the ever-present danger to the patient of viral and/or bacterial contamination in all transfusions of whole blood and red cells derived from it.
Thus, there is a recognized need for a substance that is useful for oxygen carriage and delivery under normal environmental conditions and that incorporates the following features. Ideally, the substance shall carry and deliver oxygen to devices, organs and tissues such that normal oxygen tensions may be maintained in these environments. The substance shall be non-antigenic and non-pyrogenic (i.e. less than 0.25 EU/mL). The substance shall be free of bacterial and/or viral contamination. The substance shall be safe and non-toxic. The substance shall be miscible with blood and serum. The substance shall have viscosity, colloid and oncotic properties comparable to blood. It is desirable to have a substance that will be retained in the vascular system of the patient for a long period of time, since this will permit erythropoeisis and maturation of the patient's own red cells. Furthermore, the substance shall not interfere with or hinder erythropoeisis.
It has been recognized that the natural, mammalian protein for oxygen-carriage and -delivery, hemoglobin, can be separated from the red blood cell wall membranes or stroma which contain the specific antigens that determine blood type and from other cell and plasma components. If such separation and isolation is effected, the resulting stroma-free hemoglobin contains no antigenic materials; thus, blood typing and matching are no longer necessary. For example, a typical preparation of stroma-free hemoglobin involves washing red blood cells to remove residual plasma and cell debris, lysing the red cells to release hemoglobin, and filtering and ultrafiltering the hemoglobin to separate it from contaminants. K. Bonhard, B. Eichentopf, and N. Kothe, "Process for Obtaining Hepatitis-Safe, Sterile Hemoglobin Solutions Free of Pyrogens and Stroma," U.S. Pat. No. 4,439,357, N. Kothe and B. Eichentopf, "Method of Preparing Highly Purified, Stroma-Free, Non-Hepatitic Human-Animal Hemoglobin Solutions, U.S. Pat. No. 4,526,715. The process for isolating and purifying the stroma-free hemoglobin incorporates process steps to eliminate bacterial and viral contamination. U.S. Pat. Nos. 4,598,064 and 4,600,531 (hereby incorporated by reference).
However, stroma-free hemoglobin does not meet the substance-suitability criteria defined above. For example, although it is known that stroma-free hemoglobin is capable of carrying oxygen (S. F. Rabiner et al., J. Exp. Med., Vol. 126, p. 1142, 1967.), in the absence of specific additional substances known as effectors, stroma-free hemoglobin has too high an affinity for oxygen to be useful. As a result, stroma-free hemoglobin cannot maintain normal oxygen tensions in organs and tissues. Furthermore, in its natural form, stroma-free hemoglobin is a tetrameric aggregate (molecular weight 64,500) made up of a pair of dimer-aggregates (molecular weight 32,250), each of which consists of one alpha-protein chain and one beta-protein chain. The dimer-aggregates are not held together by any covalent bond. Following infusion of stroma-free hemoglobin, this protein naturally breaks down into these pairs of dimer-aggregates, which do not deliver oxygen. The dimers are sufficiently small to be removed by filtration through the kidney and excreted in the urine. Studies have shown that the retention half-life of stroma-free hemoglobin or its breakdown dimers in the circulation is approximately two hours, i.e., the concentration is reduced by one-half every two hours. This period is far shorter than the time required for regeneration and maturation of the red blood cells in the bone marrow. Thus, stroma-free hemoglobin becomes increasingly ineffective with the passage of time. Moreover, the stroma-free hemoglobin breakdown is so rapid that the dimers accumulate in the kidney and other organs and cause damage to these organs. As a consequence, stroma-free hemoglobin may lack the clinical safety that is required of an oxygen-carrying substance. S. L. Baker and E. C. Dodds, Brit. J. Exp. Pathol. 6: 247, 1925. Taken together, all of the findings indicate that without crosslinking, tetrameric hemoglobin is unsuitable as a vehicle for a long-term delivery of oxygen to the tissue.
A number of modified hemoglobins that address some of the shortcomings of stroma-free hemoglobin are recognized. The known modification methods include various means for intramolecular crosslinking of stroma-free hemoglobin; for intermolecular crosslinking of stroma-free hemoglobin with low-molecular weight agents; for intra- and intermolecular crosslinking of stroma-free hemoglobin with low molecular weight agents; and for coupling of stroma-free hemoglobin to other polymers.
Methods for intramolecular crosslinking of stroma-free hemoglobin are known in the art. (U.S. Pat. Nos. 4,584,130, 4,598,064 and 4,600,531). For example, one of these modified hemoglobins, diaspirin crosslinked hemoglobin, is prepared by allowing stroma-free hemoglobin to react with bis(3,5-dibromosalicyl) fumarate in the presence of 2,3-diphosphoglycerate, inositol hexaphosphate or inositol hexasulfate (U.S. Pat. Nos. 4,598,064 and 4,600,531). This treatment modifies stroma-free hemoglobin by covalently linking the lysine-99 residues on the alpha chains of the protein through a fumarate bridge. As a consequence of this intramolecular cross-linking, diaspirin crosslinked hemoglobin has an oxygen affinity equivalent to that of blood. Furthermore, diaspirin crosslinked hemoglobin (molecular weight 64,500) can no longer break down into dimers (molecular weight 32,250). Since the retention time of hemoglobin in the circulatory system increases as the molecular weight of the protein increases, the retention time of diaspirin alpha-alpha crosslinked hemoglobin is four to eight hours, two to four times that of stroma-free hemoglobin. However, this is not a sufficient length of time for utility in the treatment of acute hemorrhage, since an oxygen carrier is needed that can carry oxygen for several days when the patient has lost a considerable amount of blood.
Hemoglobin molecules have also been intermolecularly crosslinked to each other through the use of low-molecular weight crosslinking agents. In particular, K. Bonhard discloses coupling hemoglobin molecules to one another and/or to serum proteins and gelatin derivatives using dialdehydes, optimally followed by the addition of pyridoxal phosphate (U.S. Pat. No. 4,336,248). Bonson et al. disclose crosslinking with a bifunctional or polyfunctional, low-molecular weight crosslinking agent. See U.S. Pat. Nos. 4,001,401, 4,001,200, 4,053,590 and 4,061,736. Typical, known products of intermolecular crosslinking of these types have oxygen-carrying and -delivery properties that are not equivalent to blood (P.sub.50 of 18-23 for glutaraldehyde-polymerized hemoglobin as compared to P.sub.50 of 28 for whole blood). Furthermore, known products of intermolecular crosslinking by glutareldehyde are antigenic (D. H. Marks et al., Military Med. Vol. 152, p. 473, 1987).
Similarly, Mock et al. (Fed. Proc. Vol. 34, p. 1458, 1975) and Mazur (U.S. Pat. No. 3,925,344) show the use of low-molecular weight, bifunctional, crosslinking agent for the preparation of intra- and intermolecular crosslinked hemoglobin. The absence of preclinical or clinical reports on the efficacy and safety of this material, which was discovered in 1975, infers that it does not meet the suitability criteria defined above.
Hemoglobin has also been coupled to polymers through the use of low-molecular weight mediators. For example, hemoglobin has been coupled to hydroxyethylstarch (German patent offenlegungsschrift No. 2,616,086); to inulin (K. Ajisaka and Y. Iwashita, "Oxygen carrier for blood substitute", U.S. Pat. No. 4,377,512); and to dextran (J. T. F. Wong, European Patent Application 0,140,640). Similarly, hemoglobin has been coupled to itself and/or to other serum proteins and gelatin derivatives using dialdehyde (3 to 8 carbon atoms) mediators, optionally followed by addition of pyridoxal phosphate (K. Bonhard and U. Boysen, U.S. Pat. No. 4,336,248). Similarly, in U.S. Pat. No. 4,179,337, peptides and polypeptides are coupled to polymers which contain a substantially linear ethereal or carbon-carbon backbone. Polyethylene glycol and polypropylene glycol are preferred. The coupling is accomplished using 10 to 100 molar equivalents of polymer to peptide or more suitably, 15 to 50 molar equivalents of polymer to polypeptide. Coupling must be accomplished with the aid of mediators. In U.S. Pat. No. 4,301,144 (Yuji Iwashita and Katsumi Ajisaka, "Blood Substitute Containing Modified Hemoglobin") hemoglobin is modified by coupling via an amide bond between a mediator-activated, terminal group of a poly(alkylene) glycol and an amino group of hemoglobin. More recent embodiments of this technology (U.S. Pat. Nos. 4,412,989 and 4,670,417) are reported to give monomeric, dimeric and trimeric modified hemoglobins. These embodiments have P.sub.50 of 21 to 25 and half-times in the circulation of 4 to 8 hours. Furthermore, the materials are so unstable that they must be lyophilized in the presence of stabilizers in order to permit storage. All of these factors indicate that derivatives of this type do not meet the criteria described above.